Development and validation of a potent and specific inhibitor for the CLC-2 chloride channel Anna K. Kostera,b, Austin L. Reesec, Yuri Kuryshevd, Xianlan Wenb, Keri A. McKiernana, Erin E. Graya, Caiyun Wud, John R. Huguenardc,1, Merritt Madukeb,1, and J. Du Boisa,1 aDepartment of Chemistry, Stanford University, Stanford, CA 94305; bDepartment of Molecular & Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305; cDepartment of Neurology & Neurological Sciences, Stanford University School of Medicine, Stanford, CA 94305; and dCharles River Laboratories Cleveland, Inc., Cleveland, OH 44128 Edited by Richard W. Aldrich, The University of Texas at Austin, Austin, TX, and approved October 22, 2020 (received for review May 21, 2020) CLC-2 is a voltage-gated chloride channel that is widely expressed that accompany from-birth genetic deletion of CLC-2 complicate in mammalian tissues. In the central nervous system, CLC-2 ap- phenotypic analysis. A potent, selective chemical reagent tar- pears in neurons and glia. Studies to define how this channel con- geting CLC-2 could inhibit channel function rapidly and re- tributes to normal and pathophysiological function in the central versibly, a decided advantage over genetic (irreversible) methods – nervous system raise questions that remain unresolved, in part for investigating channel physiology. Available Cl channel in- due to the absence of precise pharmacological tools for modulat- hibitors (39), however, suffer from low potency, with mid- – ing CLC-2 activity. Herein, we describe the development and opti- micromolar to millimolar concentrations required to inhibit Cl – mization of AK-42, a specific small-molecule inhibitor of CLC-2 with current, and lack specificity against different Cl channel sub- = ± nanomolar potency (IC50 17 1 nM). AK-42 displays unprece- types (1, 40–42). As such, the usefulness of these tool compounds dented selectivity (>1,000-fold) over CLC-1, the closest CLC-2 ho- for cellular and organismal studies is severely limited. For CLCs, molog, and exhibits no off-target engagement against a panel of the most potent and selective small-molecule inhibitors (IC50 61 common channels, receptors, and transporters expressed in ∼0.5 μM) are those targeting CLC-Ka, one of two CLC homo- brain tissue. Computational docking, validated by mutagenesis logs expressed in the kidney (43–46). For CLC-2, a peptide toxin, and kinetic studies, indicates that AK-42 binds to an extracellular GaTx2, has been identified as a selective inhibitor of CLC-2 at vestibule above the channel pore. In electrophysiological record- concentrations of ∼20 pM (47); however, reduction in peak cur- PHARMACOLOGY ings of mouse CA1 hippocampal pyramidal neurons, AK-42 acutely rent saturates at ∼50% with GaTx2 application, thus restricting and reversibly inhibits CLC-2 currents; no effect on current is ob- use of this toxin as a pharmacological probe. All other reported served on brain slices taken from CLC-2 knockout mice. These re- CLC-2 inhibitors are not selective and require application of high sults establish AK-42 as a powerful tool for investigating CLC-2 concentrations (∼1mM)(1,2,48–51) to inhibit channel function. neurophysiology. The dearth of high-affinity, high-precision tool compounds for investigating CLC-2 physiology inspires the studies described chloride channel | CLC-2 | inhibitor herein. Through a systematic structure–activity analysis, we have developed the most potent small-molecule inhibitor known to he CLC-2 ion channel is one of nine mammalian chloride – Tchannel (CLC) homologs that comprise a family of Cl -se- Significance lective channels and transporters (1). CLC-2 is expressed in nearly every organ, including brain, heart, intestines, and lungs – The CLC-2 ion channel facilitates selective passage of Cl ions (2), and is critical for electrogenesis, homeostatic control of cell across cell membranes. In the central nervous system, CLC-2 is volume, and maintenance of ion gradients. Malfunction and expressed in both neurons and glia and is proposed to regulate dysregulation of CLC-2 underlie disparate human pathologies – electrical excitability and ion homeostasis. CLC-2 has been im- (1, 3 9). In renal tissue, CLC-2 mutations are linked to primary plicated in various central nervous system disorders, including aldosteronism, the most common cause of secondary arterial – certain types of epilepsy and leukodystrophy. Establishing a hypertension in humans (10 13). In the central nervous system causative role for CLC-2 in neuropathologies, however, has (CNS), CLC-2 mutations are found in various rare forms of been limited by the absence of selective reagents that enable leukodystrophy, characterized by leukoencephalopathy, male acute and specific channel modulation. Our studies have infertility, and secondary paroxysmal kinesigenic dyskinesia (an resulted in the identification of a highly potent, small-molecule – – episodic, involuntary movement disorder) (14 20). A phenotype inhibitor that enables specific block of CLC-2 Cl currents in reminiscent of the human disease is observed in CLC-2 genetic hippocampal brain slices. This precise molecular tool should knockout mice, which exhibit severe retinal degeneration/blind- enable future efforts to identify and treat CLC-2–related ness, infertility, and vacuolization of myelin in brain tissue disease. (21–23). Additionally, CLC-2 activity influences neuronal excit- ability, as indicated by in vitro electrophysiology recordings of Author contributions: A.K.K., A.L.R., Y.K., J.R.H., M.M., and J.D.B. designed research; − − neurons from wild-type and Clcn2 / knockout animals (24–30). A.K.K., A.L.R., Y.K., X.W., K.A.M., E.E.G., and C.W. performed research; A.K.K. and The influence of CLC-2 on electrical excitability in inhibitory E.E.G. contributed new reagents/analytic tools; A.K.K., A.L.R., X.W., J.R.H., M.M., and J.D.B. analyzed data; and A.K.K., M.M., and J.D.B. wrote the paper. signaling pathways may play a role in idiopathic generalized – Competing interest statement: A.K.K., J.D.B., and M.M. have filed for a patent “Compo- epilepsies (31 37); however, a direct causal link between CLC-2 sitions and Methods to Modulate Chloride Ion Channel Activity,” USSN 16/449,021 from mutations and epilepsy has not been definitively established (38). the U.S. Patent & Trademark Office, January 16, 2020. These findings motivate subsequent research efforts to gain a This article is a PNAS Direct Submission. deeper understanding of CLC-2 function in the CNS. Published under the PNAS license. Unraveling the details of CLC-2 physiology is challenged, in 1To whom correspondence may be addressed. Email: [email protected], part, by the absence of selective chemical reagents for modu- [email protected], or [email protected]. lating channel activity. While mouse knockout models have This article contains supporting information online at https://www.pnas.org/lookup/suppl/ provided important insights into CLC-2 function, compensatory doi:10.1073/pnas.2009977117/-/DCSupplemental. changes in gene expression and developmental abnormalities www.pnas.org/cgi/doi/10.1073/pnas.2009977117 PNAS Latest Articles | 1of11 Downloaded by guest on September 24, 2021 date for any member of the CLC protein family. This compound, which a -OMe, isopropenyl, -Et, or -CF3 group was introduced at AK-42, has an IC50 of 17 ± 1 nM against human CLC-2 and C3 (AK-13, AK-14, AK-16, and AK-17, respectively). With the displays ∼10,000× greater potency toward CLC-2 compared to exception of AK-17, these ligands were nearly twofold more the most closely related CLC homolog, CLC-1. In addition, AK- potent than MCFA. In a second round of SAR, incorporation of 42 is specific for CLC-2 over a diverse panel of 61 CNS receptors, sterically larger groups at C3, which included -OBn and c channels, and transporters. We have validated the suitability of -OCH2 Hx (AK-24 and AK-26, respectively), resulted in an ad- AK-42 for physiological studies of the CNS by performing brain ditional boost in potency. Among these derivatives, AK-24 was slice recordings on both wild-type and CLC-2 knockout mice. identified as the most effective inhibitor with an IC50 of 0.6 μM. Computational and mutagenesis studies identify an AK-42 binding We next evaluated the influence of the MCFA carboxylate site on the extracellular side of the channel pore, laying the group on ligand binding (Fig. 1, Table 1, purple). This study groundwork for understanding the mechanistic underpinnings of included moving the position of the carboxylate from C1′ to C5′ channel inhibition and for further development of related tools. and C6′ on the eastern ring (AK-20 and AK-19, respectively) and The availability of AK-42 as a potent, highly selective reagent for replacing the carboxylate unit with other charged substituents, – 2– – – pharmacological knockout of CLC-2 should greatly accelerate including -OSO3 ,-PO3 ,-PO2(OEt) , -tetrazolate, and -CH2CO2 physiological studies of this channel. (AK-9, AK-34, AK-35, AK-18, and diclofenac, respectively). A neutral analog of MCFA, AK-36, in which the carboxylate group Results was converted to a primary carboxamide, was also prepared. All Screening for New CLC-2 Inhibitors. Using the IonWorks Barracuda eight compounds displayed reduced potency relative to MCFA. (IWB) automated patch-clamp platform, we performed a fo- From these data, the efficacy of carboxamide AK-36 is perhaps cused screen of Food and Drug Administration-approved drug most surprising, as this derivative is only approximately threefold molecules against human CLC-2 stably expressed in Chinese less potent than MCFA despite lacking the anionic charge. This hamster ovary (CHO) cells and identified meclofenamate result indicates that ligand–protein binding is not purely dictated (MCFA) as a “hit” compound (Fig. 1, SI Appendix, Fig. S1 and by charge–charge interactions. Table S1, and Dataset S1).